analytical approach for nanomaterials characterization in

Analytical approach for nanomaterials characterization in the nanosafety context

Federico BENETTIECSIN LAB – ECAMRICERT SRL a Mérieux NutriSciences Company

EcamRicert-ECSIN, a Mérieux NutriSciences company

2

ECSIN

EUROPEAN CENTER

FOR THE SUSTAINABLE

IMPACT OF

NANOTECHNOLOGY

SUSTAINABLE DEVELOPMENT OF NANOTECHNOLOGY

Efficacy Safety

Regulation

3

Nanomaterials definition

The EU adopted a definition of a nanomaterial in 2011 - Recommendation on the definition of a nanomaterial (2011/696/EU)

Source: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32011H0696

A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as anagglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is inthe size range 1 nm - 100 nm.

In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number sizedistribution threshold of 50 % may be replaced by a threshold between 1 and 50 %.

The Recommendation 2011 applies to “natural, incidental or manufactured” nanomaterials as below described:

▪ Natural materials occur in the environment without human intervention, as for example volcanic ashes, clay minerals

▪ Manufactured materials usually refers to materials made for a specific purpose

▪ Incidental means materials resulting from a human-induced process with a purpose other than producing nanomaterials, as for

example combustion processes

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Nanomaterials use - exposure

PRODUCT

NANOMATERIAL

INTENTIONAL

Aware

Improving product properties

NON-INTENTIONAL

Unaware (accidental)

Production processes

Unaware

Additives or ingredients

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Nanomaterial identification & characterization

NANOMATERIALS

BIOLOGICAL

PROPERTIESEfficacy Safety

• Novel foods Reg. 2015/2283

• Food additives Reg. 2008/1333

• Medical devices prop. COD 2012/0266

Intentional useUnintentional use

PHYSICO-CHEMICAL

PROPERTIESIDENTIFICATION CHARACTERIZATION

Functionality

REGULATORY

COMPLIANCEPRODUCTDEVELOPMENT

CHEMICAL-PHYSICAL CHARACTERIZATION, WHEN & WHY


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Tech

niq

ue

TE

M

DLS

A4F

spIC

P-M

S

ICP

- M

S

ED

X

BE

T

Dete

rmin

able

for

so

me n

an

om

ate

ria

ls

Not d

ete

rmin

able

Property of the nanomaterial

Size/Size distribution

2D projected geometrical

Dete

rmin

able

Hydrodynamic

Mass equivalent

Agglomeration/aggregation state 1 1

Shape

Spatial distribution

Concentration 2 3 2/3 2

Chemical composition

Inorganic 2 2 3 3 2

Crystalline structure

Surface area

Target performance

Direct analysis of solids* W

ell

fulfill

ed

Part

ially

fu

lfill

ed

Scarc

ely

fulfi

lled

Minimum sample preparation

Applicable to complex matrices

Adequate size range (1-100 nm) 5 4

Wide concentration range 3

Minimum preliminary information

Operatively simple

Cheap

* analysis of liquids is always assumed 1 assumptions or complementary information needed 2 requires coupled/combined technique 3 depends on matrix interference 4 depends on the coupled technique 5 depends on sample concentration

LEGEND

TEM – transmission electron microscopy;

DLS – dynamic light scattering;

A4F – asymmetric flow field-flow fractionation;

spICP-MS – single particle ICP-MS;

ICP-MS – inductively coupled plasma mass spectrometry;

EDX - Energy-dispersive X-ray spectroscopy;

BET - Brunauer-Emmett-Teller surface area analysis.

Main techniques to study nanomaterials summary

The multi-technique approach for testing nanomaterials approach for detection and characterisation:

1. Qualitative screening

2. Size distribution and morphology

3. Chemical Identification

4. Quantification

5. Risk assessment (if needed)

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Chemical composition, quantification and purity

(spICP-MS, EDX, HPLC)

Morphology

(TEM)

Size distribution

(TEM, DLS, AF4, SP-ICP-MS)

Surface area

(BET)

Superficial charge

(DLS, ζ potential)

Solubility and stability

(SP-ICP-MS, HPLC, DLS)


THE MULTI-TECHNIQUE APPROACH

TO NANOMATERIALS TESTING


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THE MULTI-TECHNIQUE APPROACH TO NANOMATERIALS TESTING

▪ Is sample homogeneous or heterogeneous?

▪ Is the statistics adequate?

▪ Is any alteration taking place or artefact formation? (size, morphology, aspect ratio)

▪ Batch-to-batch variation in the manufacturing process

▪ Ageing effects during storing (agglomeration, aggregation, sedimentation)

▪ False positive/ false negative?

Nanomaterial identification

PARTICLE SIZE – DATA INFORMATION

▪ Data on primary and secondary (agglomerates and aggregates) PARTICLE SIZE, NUMBER-BASED SIZE DISTRIBUTION and MASS-

BASED SIZE DISTRIBUTION of the material should be provided as measured by more than one independent technique, one

being electron microscopy (EM).

▪ Data should be provided both as MEDIAN PARTICLE DIAMETER (x50 in nm), with an indication of the width of the distribution (e.g.

standard deviation, in nm) and with an estimate of the uncertainty of the median diameter (expanded uncertainty, confidence level

95%, in nm). Together with the size distribution information on the lower and upper cut-off limits for the calculation of the relative

amount of particles has to be provided.

▪ If other techniques are used, a conversion to the number-based size distribution must also be provided, including information on

the algorithms used for conversion and the associated uncertainty.

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Nanomaterial identification: Chemical Identification (EDX)

CASE STUDY


540 x 15000 x 150000 x

Low and medium magnification:

aggregates 0.1÷10 μm

No free particles

High magnification:

all aggregates are made up of

10.5 nm primary particles

I T I S AN A N O M A T E R I A L

I T ’ S N O T AN A N O M A T E R I A L


VOLUME SPECIFIC SURFACE AREA – DATA INFORMATION

▪ In compliance with the definition, nanomaterials can be classified on the basis of the specific surface area by volume.

▪ A material should be considered as falling under the definition if the specific surface area by volume of the material is greater than

60 m2/cm3.

▪ However, a material which, based on its number size distribution, is a nanomaterial should be considered as complying with the

definition even if the material has a specific surface area lower than 60 m2/cm3.

(2011/696/EU)

NANOSTRUCTURED MATERIALS

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For the equivalent volume, the smaller

the size the greater the specific surface

area

Area= 6x1 m2 = 6 m2

Area= 6x(1/2 m)2 x x8 = 12 m2

Area= 6x(1/3 m)2

x27 = 18 m2

▪ Nanoparticles, aggregates and agglomerates have high specific surface area

▪ Non-nanomaterials in terms of size may fall within the definition due to their high porosity (nanostructured)

Nanomaterial specific surface area determined by BET (Brunauer-Emmett-Teller) method

SPECIFIC SURFACE AREA

ECSIN LABpart of Mérieux NutriSciences Group

BET ACCREDITED

Nanomaterial characterization during exposureType of nanomaterial application

ingredient, additive, pesticide, food contact material

Quantify

migration/transferring

Identify (possibly quantify)

engineered nanomaterials or their

(non-nano) degradation forms in

food, cattle feed

Identify (possibly quantify)

engineered nanomaterials or their

(non-nano) degradation forms in

food simulants, food, cattle feed

Presence due to

migration/transferring

NANOFORMS MUST BE

INCLUDED IN THE PROCEDURE

OF EXPOSURE ASSESSMENT

NANOFORMS CAN BE EXCLUDED

FROM THE PROCEDURE OF

EXPOSURE ASSESSMENT

Are there still engineered

nanomaterials?

Directly added

YES NO

Guidance on the risk assessment of the application of nanoscience and nanotechnologies in

the food and feed chain EFSA Journal 2018;16(7):5327

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Example: Risk assessment of nanomaterials in food and feed

Two important questions:

• Does the material quickly degrade in in vitro digestive

tract conditions?

• Is there a potential for the material to be biopersistent

and/or hazardous?

Guidance on the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain

EFSA Journal 2018;16(7):5327


Advantages

▪ Size distribution

▪ Wide size range (0.3 nm – 10 µm)

▪ High concentration (40% w/v)

▪ Fast & cheap

Disadvantages

▪ Hydrodynamic size

▪ No morphological information

▪ Number-based size distribution calculated

▪ Low sensitivity

▪ Not suitable for polydisperse samples

Dynamic Light Scattering (DLS)PARTICLE SIZE ANALYSIS ACCORDING TO ISO 22412

Hydrodynamic diameter (Dh) - equivalent sphere with diffusion coefficient as analyzed particles

Dh Dh DhDh DLS

TEM

10

10

10 si

gnal

inte

nsi

ty, c

ps

10

100

sign

al in

ten

sity

, cp

s

10

300

100000

Integration time 1000 ms



Integration time 0.1 ms




Integration time 0.1 ms

Standard

Single particle

ADVANTAGES

▪ Number- and mass-based particles concentration

▪ Number- and mass-based particles size distribution

▪ Ionic fraction

DISAVANTAGES

▪ Single element analysis

▪ Element dependent performances

spICP-MS - single-particles ICP-MS

Quantitative determination of elements according to ISO/TS 19590 «Nanotechnologies - size distribution and concentration

inorganic nanoparticles in aqueous media via single particle inductively coupled plasma mass spectrometry»:

Nanomaterial characterization

A4F – MALS – ICP-MS

▪ separative capabilities

▪ chemical characterization

▪ mass concentration

Nanomaterial characterization

ADVANTAGES

▪ Sensibility

▪ Specificity

▪ Quantitative

▪ Dimensional information

◼ Testing of nanoparticles are not like testing of any other contaminants: no official methods and high competencesneeded

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Conclusions

◼ Data interpretation of needs and results require support of expertise

◼ Lab malpractices may create artifacts or destroying pristine material resulting in false positive or false negative results

◼ Characterization of a nanomaterial requires complementary methodologies and a tiered-approach

◼ Unintentional presence of nanomaterials poses great attention in evaluating the safety of products

THANK YOU

FOR YOUR ATTENTION

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